ABSTRACTInsulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

fig6: Comparison of the effects of insulin, NO, and thrombin on the activation of αIIbβ3 integrin and platelet α-granule secretion. Representative FACS® analysis showing the effects of stimulation (10 min) with either solvent (CTL), insulin (Ins, 1 μmol/L), DETA NONOate (NO, 1 μmol/L), or thrombin (Thr, 0.1 U/ml) on the surface expression of activated αIIbβ3 integrin (PAC1) or P-selectin. Identical results were obtained in four additional experiments. The dashed line represents the population of unlabeled platelets.

Mentions:
Neither insulin nor DETA NONOate activated αIIbβ3 integrin, as assessed using a FITC-conjugated PAC1 antibody that recognizes only the activated conformation of the αIIbβ3 complex (Fig. 6). Moreover, neither stimulus elicited the cell surface expression of P-selectin. These observations contrasted markedly with the effects of thrombin, which both activated αIIbβ3 integrin and elicited the cell surface expression of P-selectin (Fig. 6). Insulin also failed to effect either the release of the α-granule marker β-thromboglobulin or the generation of thromboxane A2 (not depicted).

fig6: Comparison of the effects of insulin, NO, and thrombin on the activation of αIIbβ3 integrin and platelet α-granule secretion. Representative FACS® analysis showing the effects of stimulation (10 min) with either solvent (CTL), insulin (Ins, 1 μmol/L), DETA NONOate (NO, 1 μmol/L), or thrombin (Thr, 0.1 U/ml) on the surface expression of activated αIIbβ3 integrin (PAC1) or P-selectin. Identical results were obtained in four additional experiments. The dashed line represents the population of unlabeled platelets.

Mentions:
Neither insulin nor DETA NONOate activated αIIbβ3 integrin, as assessed using a FITC-conjugated PAC1 antibody that recognizes only the activated conformation of the αIIbβ3 complex (Fig. 6). Moreover, neither stimulus elicited the cell surface expression of P-selectin. These observations contrasted markedly with the effects of thrombin, which both activated αIIbβ3 integrin and elicited the cell surface expression of P-selectin (Fig. 6). Insulin also failed to effect either the release of the α-granule marker β-thromboglobulin or the generation of thromboxane A2 (not depicted).

ABSTRACTInsulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.